The tryptophan synthase alpha2beta2 multienzyme complex is an excellent model system for investigating enzyme mechanism, protein-protein interaction, the allosteric mechanism and metabolite channeling. Correlations between the functional properties of wild type and mutant forms of tryptophan synthase and the three-dimensional structure have produced recent important results: (1) Mechanism of pyridoxal phosphate- dependent reactions. Lysine-87 forms a Schiff base with the pyridoxal phosphate coenzyme in active site of the wild type beta subunit. Changing lysine-87 to threonine produces an inactive mutant alpha2beta2 complex that forms enzyme-substrate intermediates very slowly. Spectroscopic and kinetic studies of this mutant enzyme show that lysine-87 serves critical roles in transamination, catalysis, and product release. Two new crystallographic structures of this mutant enzyme containing bound L-serine or L-tryptophan reveal important information about the substrate binding site of the beta subunit. (2) Mechanism of subunit communication. Studies using site-directed mutagenesis and limited proteolysis provide evidence that a flexible loop in the alpha subunit is important for ligand binding and for communicating the effects of ligand binding from the alpha subunit to the beta subunit in the alpha2beta2 complex. A residue in the alpha subunit loop (threonine-183) plays a critical role in modulating the enzymatic activity of the beta subunit in the alpha2beta2 complex. Two alpha subunit residues that are located in the interaction site between the alpha and beta subunits (proline-57 and proline-132) are important for mutual subunit interaction activation. Mutation of glutamate-49, aspartate-60, or glycine-51 in the alpha subunit inhibits the transition of the alpha subunit from open to a closed form and alters the kinetics of metabolite channeling. (3) Conformational states of the beta subunit. Our finding that the dimeric wild type beta subunit is only 50% inactivated by beta-chloro-L-alanine can be explained by the presence of two conformers of the subunit in solution: one conformer is rapidly inactivated by beta- chloro-L-alanine and the other conformer is not activated. This putative mechanism is supported by investigations using steady-state kinetics and spectroscopic and electrophoretic methods.